Molecular Cloning And Spatio-temporal Expression Of FUT2-like Gene In The Oyster (Crassostrea Gigas)

Norovirus(NoV) is the leading cause of acute viral gastroenteritis worldwide,usually causes serious public health and food safety events. Histo-blood groupantigens (HBGAs) are recognized as receptors of NoV, α-1,2-fucosyltransferase(FUT2) is one of key enzymes for HGBAs biosynthesis pathway. It is reported thattype A-like HBGA present in oyster (Crassostrea gigas) gastrointestinal cells, whichinduces specific accumulation of NoV in oysters. This research is based on theNational Natural Science Foundation of China. FUT2-like protein which is the keyenzyme of A-like HBGA in oyster is studied. In this work, molecular cloning andexpression of gene in the oyster is studied.A-like HBGA is existed in oyster, but studies involving the oyster or otheraquatic animal FUT2have rarely been reported. In this study, molecular cloning andexpression analysis of FUT2-like gene were performed in the oyster. The GenBankaccession number was KJ184342. As a result, the FUT2-like cDNA sequence is1941bp, which contains a5’-untranslated region (UTR) of180bp, an open readingframe (ORF) of1086bp encodinga proteinof361amino acids, and a3’-untranslatedregion (UTR) of675bp. The tissue expression distribution pattern of FUT2-like genewas examined in the5tissues of oyster. Analysis of molecular evolution showedthat the oyster FUT2-like gene was similar to the mammalian FUT2gene such as Musmusculus.First the codon was optimized for Escherichia coli. Then the oyster FUT2-likegene amplification was complicated, pRSET A-mof expression vector wasconstructed, to express in E.coli. As a result, the protein expression condition was1.0mmol/L IPTG,37℃induced4hours. Expressed protein was identified bySDS-PAGE and western blotting, using6×His-Tag monoclonal antibody and human FUT2monoclonal antibody. The result was that the relative molecular mass ofexpressed protein was48kDa, which was corresponded to theoretical value (41.62kDa).For oyster FUT2-like gene, the real-time fluorescent quantitative PCR detectionmethod was established, designed primers, using SYBR Green, referenced β-actin,based on Delta-Delta CTrelative quantitative method, to research the spatio-temporalexpression. As a result, among the five different organizations the expressionquantity in June was the lowest. In February and March, the expression quantity wason a declining curve. Between June and December, the expression quantityappeared the trend of the first increase after the fall. Overall, between July andNovember, the expression quantity generally was on the high side. The resultsshowed that the FUT2-like mRNA was predominantly expressed in labial palp, andless expressed in other four organizations which is digestive tissues, adductor muscle,mantle and gills. The expression quantity in the four organizations was nosignificant difference (P<0.01). The established ELISA detection method was usedto study the expression pattern of A-like HBGA. Every month, A-like HBGA in fivedifferent organizations was detected, such as digestive tissues, adductor muscle,mantle, labial palp, gills. As a result, the differences were not obvious in differentorganizations, and the content was high in August to May the following year. Itscontent was showed a trend of decline from January to March, a trend of rising fromMarch to May, and generally an upward trend from June to November. The contentwas higher from August to November. The research confirmed that thespatio-temporal expression of FUT2-like gene in oyster had a relationship with A-likeHBGA.This study explores the oyster specific molecular mechanism on enrichment NoV,to provide research foundation for scientific prevention and control outbreaks offood-borne outbreak, and guarantee the oyster breeding industry. And this will provide the references about the shellfish accumulating toxic and harmful substanceson molecular mechanism.